Bubbles being introduced to media tubing

Experiments
1

I’m getting a lot of bubbles in my tubing. Some background:

  • I fill the vials using the evolver tubing by pumping for around a minute. There will be no bubbles at this time.
  • I’ll inoculate with a small amount of bacteria, and have a turbidostat mode with an upper threshold of say 0.4
  • Overnight, if there are no pumping events, bubbles get introduced into the system. There are bubbles on both sides of the media input lines ( from media and to vial).
    -I’m currently using massive carboys for media. These are place on the ground. But I’ve also encountered bubbles using a 1 L media bottle on the benchtop level.
  • One media carboy is attached to 5 vials.

This bubbles can be a bit problematic. I have phage + bacteria in my system in a turbidostat mode. Sometimes there are long periods - like 50 hours ( where the phage has killed bacteria, and bacteria is evolving) with no pumping events. Bubbles can be introduced here. When a pumping event is triggered, it will just pump air instead of media.
I know the turbidostat mode has code that protects the system somewhat from bubbles, triggering additional dilutions if needed. However, multiple dilutions are not ideal and can lead to diluting past the lower threshold. In a chemostat regime, bubbles would be more problematic as the dilution rate would in fact be much lower than expected.

Are there any ways to reduce the amount of bubbles introduced into the system? Does the peristaltic pump introduce some bubbles?

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From my experience, bubbles are formed from loose connections in your lines, I do not see this with normal operation of the peristaltic pumps.

Secondary question, do your media bottles have a vent? It might have a difficult time pumping the liquid if it is sealed with a cork.

Brandon

3

For anything diluting somewhat frequently (normal turbidostat or chemostat), I’d agree with Brandon, the number of bubbles in your lines would be unusual. Check that connections are tight and that the media bottles are vented to prevent vacuum.

For the specific case that you described, with 50 hours between dilution events, with media bottles on the floor as you have them, I could imagine bubbles building up over time. We know the tubing is not gas impermeable and that media/bleach left in lines for weeks can dry out, so its possible air gets in there somehow, or that media is being pulled down by gravity? I am still skeptical though, because those photos have a lot more bubbles (especially large ones) than I would expect over only 50 hours.

If you notice you’ve got a line with bubbles that would cause problems, you could always disconnect that media line from the vial and flush the line into another container manually using the electron app before reconnecting. Be careful not to contaminate at this step.

4

Update: I put the media carboys about 1m above the eVOLVER- it made an enormous difference, no bubbles so far!

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